Lipopolysaccharide (LPS) is a major component of the outer membrane of gram negative bacteria. The endotoxic component of LPS is the lipid A portion. It comprises 1,6-linked D-glucosamine residues that are substituted with up to six acyl chains and a core polysaccharide structure to which additional polysaccharide repeating units may be attached. Endotoxin is a potent activator of the innate immune system at low doses while at higher doses endotoxin induces a number of other physical reactions including septic shock and death (Heine et al., 2001). Contamination of therapeutic products with endotoxins is therefore a primary concern for the manufacturers of such products.
Many recombinant proteins are produced in the gram negative bacteria Escherichia coli. The removal of LPS from these recombinant proteins can be a complicated but essential process especially if the proteins are destined for therapeutic uses. Many different processes have been developed for the removal of LPS from proteins based on the unique molecular properties of the endotoxin molecules. These include LPS affinity resins, two-phase extractions, ultrafiltration, hydrophobic interaction chromatography, ion exchange chromatography, and membrane adsorbers (reviewed by Petsch and Anspach, 2000). These procedures have varying degrees of success in the separation of LPS from proteins, which in a large part is dependent on the properties of the protein of interest.
Often, during the production of recombinant proteins, difficulties in the separation of LPS from proteins are encountered due to protein-LPS interactions. For example, and not by way of limitation, in recombinant proteins produced from an E. coli expression system. Several of the published procedures for the separation of LPS from proteins have been investigated, including, but not necessarily limited to, denaturing hydrophobic interaction chromatography (Wilson et al., 2001) and the use of ethanol, isopropanol (Franken et al., 2000), or detergent (Fiske et al., 2001) washes while the protein was immobilized on ion-exchange chromatographic media.
Some experiments have shown that alcohol and detergent washes during ion exchange chromatography are effective in reducing the protein associated LPS levels while poor separation of LPS from the proteins was obtained by the denaturing HIC procedure. The detergents (Zwittergent® 3-12 or 3-14) were shown to be more effective washing agents than the alcohols. Improved LPS clearance was also been achieved while the LPS-protein complexes were bound to a cation exchange resin as opposed to an anion exchange resin but the washing procedures used to remove LPS were effective on both matrices. When the wash procedure is performed on a cation exchanger, once the LPS-protein interactions have been disrupted the LPS should be washed out of the column while the protein is retained. During anion exchange chromatography, the LPS, being negatively charged at most pHs, remains bound to the resin along with the protein. Even though the alcohol and detergent washes were successful at reducing the levels of LPS in the LPS-protein complexes, scaling up and implementing any of these procedures in a manufacturing setting would not be practical. The concentrations of ethanol and isopropanol required to effectively reduce the LPS levels of the LPS binding proteins were greater than 50% (v/v). At these concentrations, these solutions are considered flammable liquids and as such impose many safety and operational restrictions.
The detergents, even though very effective at reducing LPS levels, are relatively expensive and would add significant cost to a manufacturing process and may affect the bioactivity of the protein of interest. Accordingly, Alternative chemicals are desired that could safely and cost effectively be used in place of the alcohols or detergents as washing agents for the separation of LPS from proteins during chromatographic unit operations. Ideally, these chemicals would be relatively inexpensive, well defined chemically, present minimal safety issues, and have minimal impact on the bioactivity of the protein in question when implemented into a process.